Characteristics of Protein-Kinase-C- and ADP-Ribosylation-Factor-Stimulated Phospholipase D Activities in Human Embryonic Kidney Cells
| Autor: | Karl Jakobs, Martina Schmidt, Andreas Grannass, Eva Tapp, Friederike Wahn, Ulrich Rümenapp |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Guanine
GTP' ADP ribosylation factor Guanosine Digitonin Biology Kidney Biochemistry Permeability Cell Line chemistry.chemical_compound GTP-Binding Proteins Phospholipase D Humans Phosphatidylinositol Protein Kinase C Protein kinase C ADP-Ribosylation Factors GTPase-Activating Proteins HEK 293 cells Neomycin Staurosporine Molecular biology Isoenzymes chemistry Guanosine 5'-O-(3-Thiotriphosphate) Phorbol Tetradecanoylphorbol Acetate ADP-Ribosylation Factor 1 |
| Zdroj: | European Journal of Biochemistry. 248:407-414 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1997.00407.x |
| Popis: | Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbol-ester-activated protein kinase C (PKC) and by membrane receptors, the latter apparently acting via the GTP-binding proteins, ADP-ribosylation factor (ARF) and Rho. In the present study, performed in cell-free preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In digitonin-permeabilized HEK cells, prelabeled with [3H]oleic acid, GTP[S] and PMA caused an approximately threefold concentration-dependent increase in the formation of [3H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD cofactor, phosphatidylinositol 4,5-bisphosphate, decreased basal and GTP[S]- or PMA-stimulated PLD activities with similar sensitivity. GDP and its analogue, guanosine 5'-O-[beta-thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the nonselective PKC inhibitor, staurosporine, but not vice versa. PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cytosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine-nucleotide-exchange factor for ARF1. potentiated the GTP[S]-stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 and bisindolylmaleimide I. Immunoblot analysis demonstrated that both conventional PKC (alpha, beta, gamma) and atypical PKC isozymes (zeta, tau) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation-dependent mechanism involving membrane-associated PKC isozymes but not ARF proteins, the major targets of GTP[S]' action. |
| Databáze: | OpenAIRE |
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